The invention relates generally to screening methods involving use of metabolic oxidation-reduction indicator dyes for identifying antimicrobial agents.
Infectious diseases can be caused by a number of organisms, including bacteria, fungi, protozoans and other parasites, and viruses. Bacteria as a group generally include gram-negative bacteria, gram-positive bacteria, spirochetes, rickettsiae, mycoplasmas, mycobacteria and actinomycetes. Resistance of bacteria and other pathogenic organisms to antimicrobial agents is an increasingly troublesome problem. The accelerating development of antibiotic-resistant bacteria, intensified by the widespread use of antibiotics in farm animals and overprescription of antibiotics by physicians, has been accompanied by declining research into new antibiotics with different modes of action. [Science, 264: 360-374 (1994).]
Antibacterial agents can be broadly classified based on chemical structure and proposed mechanism of action, and major groups include the following: (1) the xcex2-lactams, including the penicillins, cephalosporins and monobactams; (2) the aminoglycosides, e.g., gentamicin, tobramycin, netilmycin, and amikacin; (3) the tetracyclines; (4) the sulfonamides and trimethoprim; (5) the fluoroquinolones, e.g., ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7) the macrolides, which include for example, erythromycin, azithromycin, and clarithromycin; and (8) other antibiotics, e.g., the polymyxins, chloramphenicol and the lincosamides.
Antibiotics accomplish their anti-bacterial effect through several mechanisms of action which can be generally grouped as follows: (1) agents acting on the bacterial cell wall such as bacitracin, the cephalosporins, cycloserine, fosfomycin, the penicillins, ristocetin, and vancomycin; (2) agents affecting the cell membrane or exerting a detergent effect, such as colistin, novobiocin and polymyxins; (3 ) agents affecting cellular mechanisms of replication, information transfer, and protein synthesis by their effects on ribosomes, e.g., the aminoglycosides, the tetracyclines, chloramphenicol, clindamycin, cycloheximide, fucidin, lincomycin, puromycin, rifampicin, other streptomycins, and the macrolide antibiotics such as erythromycin and oleandomycin; (4) agents affecting nucleic acid metabolism, e.g., the fluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine, griseofulvin, rifamycins; and (5) drugs affecting intermediary metabolism, such as the sulfonamides, trimethoprim, and the tuberculostatic agents isoniazid and para-aminosalicylic acid. Some agents may have more than one primary mechanism of action, especially at high concentrations. In addition, secondary changes in the structure or metabolism of the bacterial cell often occur after the primary effect of the antimicrobial drug.
Protozoa account for a major proportion of infectious diseases worldwide, but most protozoan infections occur in developing countries. Treatment of protozoan infections is hampered by a lack of effective chemotherapeutic agents, excessive toxicity of the available agents, and developing resistance to these agents.
Fungi are not only important human and animal pathogens, but they are also among the most common causes of plant disease. Fungal infections (mycoses) are becoming a major concern for a number of reasons, including the limited number of antifungal agents available, the increasing incidence of species resistant to known antifungal agents, and the growing population of immunocompromised patients at risk for opportunistic fungal infections, such as organ transplant patients, cancer patients undergoing chemotherapy, burn patients, AIDS patients, or patients with diabetic ketoacidosis. The incidence of systemic fungal infections increased 600% in teaching hospitals and 220% in non-teaching hospitals during the 1980""s. The most common clinical isolate isolated is Candida albicans (comprising about 19% of all isolates). In one study, nearly 40of all deaths from hospital-acquired infections were due to fungi. [Sternberg, Science, 266:1632-1634 (1994).].
Known antifungal agents include polyene derivatives, such as amphotericin B (including lipid or liposomal formulations thereof) and the structurally related compounds nystatin and pimaricin; flucytosile (5-fluorocytosine); azole derivatives (including ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, tioconazole, terconazole, fluconazole, itraconazole, voriconazole [Pfizer], poscaconazole [SCH56592, Schering-Plough]) and ravuconazole; allylamines-thiocarbamates (including tolnaftate, naftifine and terbinafine); griseofulvin; ciclopirox; haloprogin; echinocandins (including caspofungin [MK-0991, Merck], FK463 [Fujisawa] and VER-002 [Versicor]); nikkomycins; and sordarins. Recently discovered as antifungal agents are a class of products related to bactericidal permneability-increasing protein (BPI), described in U.S. Pat. Nos. 5,627,153, 5,858,974, 5,652,332, 5,856,438, 5,763,567 and 5,733,872, the disclosures of all of which are incorporated herein by reference.
Bactericidal/permeability-increasing protein (BPI) is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. See Elsbach, 1979, J. Biol. Chem., 254: 11000; Weiss et al., 1987, Blood 69: 652; Gray et al., 1989, J Biol. Chem. 264: 9505. The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein (SEQ ID NOS: 1 and 2) have been reported in U.S. Pat. No. 5,198,541 and FIG. 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. Recombinant human BPI holoprotein has also been produced in which valine at position 151 is specified by GTG rather than GTC, residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG) and residue 417 is alanine (specified by GCT) rather than valine (specified by GTT). An N-terminal fragment of human BPI possesses the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. (Ooi et al., 1987, .J. Bio. Chem. 262: 14891-14894). In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms and some endotoxin neutralizing activity. (Ooi et al., 1991, J. Exp. Med. 174: 649). An N-terminal BPI fragment of approximately 23 kD, referred to as rBPI23, has been produced by recombinant means and also retains anti-bacterial, including anti-endotoxin activity against gram-negative organisms (Gazzano-Santoro et al., 1992, Infect. Immun. 60: 4754-4761). An N-terminal analog designated rBPI21 (also referred to as rBPI(1- 193)ala132) has been described in U.S. Pat. No. 5,420,019.
Three separate functional domains within the recombinant 23 kD N-terminal BPI sequence have been discovered (Little et al., 1994, J. Biol. Chem. 269: 1865). These functional domains of BPI designate regions of the amino acid sequence of BPI that contributes to the total biological activity of the protein and were essentially defined by the activities of proteolytic cleavage fragments, overlapping 15-mer peptides and other synthetic peptides. Domain I is defined as the amino acid sequence of BPI comprising from about amino acid 17 to about amino acid 45. Initial peptides based on this domain were moderately active in both the inhibition of LPS-induced LAL activity and in heparin binding assays, and did not exhibit significant bactericidal activity. Domain II is defined as the amino acid sequence of BPI comprising from about amino acid 65 to about amino acid 99. Initial peptides based on this domain exhibited high LPS and heparin binding capacity and exhibited significant antibacterial activity. Domain III is defined as the amino acid sequence of BPI comprising from about amino acid 142 to about amino acid 169. Initial peptides based on this domain exhibited high LPS and heparin binding activity and exhibited surprising antimicrobial activity, including antifungal and antibacterial (including, e.g., anti-gram-positive and anti-gram-negative) activity. The biological activities of peptides derived from or based on these functional domains (i.e., functional domain peptides) may include LPS binding, LPS neutralization, heparin binding, heparin neutralization or antimicrobial activity.
BPI protein products are described to have a variety of antimicrobial activities. For example, BPI protein products are bactericidal for gram-negative bacteria, as described in U.S. Pat. Nos. 5,198,541, 5,641,874, 5,948,408, 5,980,897 and 5,523,288. International Publication No. WO 94/20130 proposes methods for treating subjects suffering from an infection (e.g. gastrointestinal) with a species from the gram-negative bacterial genus Helicobacter with BPI protein products. BPI protein products also enhance the effectiveness of antibiotic therapy in gram-negative bacterial infections, as described in U.S. Pat. Nos. 5,948,408, 5,980,897 and 5,523,288 and International Publication Nos. WO 89/01486 (PCT/US99/02700) and WO 95/08344 (PCT/US94/11255). BPI protein products are also bactericidal for gram-positive bacteria and mycoplasma, and enhance the effectiveness of antibiotics in gram-positive bacterial infections, as described in U.S. Pat. Nos. 5,578,572 and 5,783,561 and International Publication No. WO 95/19180 (PCT/US95/00656). BPI protein products exhibit antifungal activity, and enhance the activity of other antifungal agents, as described in U.S. Pat. No. 5,627,153 and International Publication No. WO 95/19179 (PCT/US95/00498), and further as described for BPI-derived peptides in U.S. Pat. No. 5,858,974, which is in turn a continuation-in-part of U.S. application Ser. No. 08/504,841 and corresponding International Publication Nos. WO 96/08509 (PCT/US95/09262) and WO 97/04008 (PCT/US96/03845), as well as in U.S. Pat. Nos. 5,733,872, 5,763,567, 5,652,332, 5,856,438 and corresponding International Publication Nos. WO 94/20532 (PCT/US94/02465) and WO 95/19372 (PCT/US94/10427). BPI protein products exhibit anti-protozoan activity, as described in U.S. Pat. Nos. 5,646,114 and . 6,013,629 and International Publication No. WO 96/01647 (PCT/US95/08624). BPI protein products exhibit anti-chlamydial activity, as described in co-owned U.S. Pat. No. 5,888,973 and WO 98/06415 (PCT/US97/13810). Finally, BPI protein products exhibit anti-mycobacterial activity, as described in co-owned, co-pending U.S. application Ser. No. 08/626,646, which is in turn a continuation of U.S. application Ser. No.08/285,803, which is in turn a continuation-in-part of U.S. application Ser. No.08/031,145 and corresponding International Publication No. WO 94/20129 (PCT/US94/02463).
Of interest to the background of the present invention are metabolic oxidation-reduction indicator dyes, which measure electron transport activity. For example, Alamar Blue(trademark), a tetrazolium based dye, is an oxidation-reduction indicator that both fluoresces and changes color in response to chemical reduction resulting from cell growth.
There continues to exist a need for novel antimicrobial agents useful for treating a variety of infections and for methods of identifying such novel compounds. Such methods ideally would provide for rapid and highly selective identification of compounds that may be structurally distinct from the major conventional antimicrobial agents.
The present invention generally provides methods for identifying antimicrobial compounds (including, for example, antifungal or antibacterial compounds) based on the discovery that a class of antimicrobial agents based on or derived from bactericidal/permeability-increasing protein (BPI) generates unique effects on fungal and bacterial cells as revealed by treatment with a metabolic oxidation-reduction indicator dye, Alamar Blue(trademark). When BPI-derived peptide compounds are employed as antifungal agents, their effects are characterized by an unexpected apparent increase in metabolic oxidation-reduction activity before or concurrently with an onset of loss or reduction of fungal cell viability at the same peptide concentration. Similarly, when rBPI21 or BPI-derived peptide compounds are employed as antibacterial agents, their effects are also charactcrized by an apparent increase in metabolic oxidation-reduction activity before or concurrently with an onset of loss or reduction of bacterial viability at the same peptide concentration.
Novel antimicrobial agents may be rapidly and selectively identified by screening candidate test compounds for replication of the characteristic apparent increase in target cell metabolic oxidation-reduction activity (relative to untreated control cells) that is produced by BPI protein products before or concurrently with the onset of loss (including reduction) of viability at the same candidate compound concentration within the tested target cell population. Sources of test compounds include, for example, libraries (including combinatorial libraries) of inorganic or organic compounds (for example, bacterial, fungal, mammalian, insect or plant products, peptides, peptidomimetics and/or organomimetics). Presently preferred standard BPI-derived peptides that are known to produce this characteristic pattern include XMP.391 (SEQ ID NO: 4) or XMP.445 (SEQ ID NO: 6).
This aspect of the invention thus contemplates a method of identifying a potential antimicrobial agent, particularly an antifungal compound, comprising the steps of: (a) contacting a target cell (e.g., a fungal cell or a bacterial cell) with a metabolic oxidation-reduction indicator dye in the presence and absence of test compound, and (b) detecting apparent increased metabolic activity in the presence of the test compound relative to metabolic activity in the absence of the test compound, before or concurrently with onset of loss or reduction of target cell viability at the same candidate compound concentration within the tested target cell population, or despite eventual loss or reduction of target cell viability. Compounds that provide this fingerprint are then selected as potential antimicrobial compounds and may undergo further testing. Any metabolic oxidation-reduction indicator dye capable of detecting metabolic activity, including mitochondrial metabolic activity, may be used; a presently preferred metabolic oxidation-reduction indicator dye is Alamar Blue(trademark) [AccuMed Int""l, Westlake, Ohio]. The eventual loss or reduction of target cell viability may be confirmed by routine culture, through use of other dyes such as propidium iodide or trypan blue, or through the metabolic oxidation-reduction indicator dye itself by monitoring dye signal over time (wherein a lack of change in dye signal indicates that the cells have died).
It is further contemplated that screening methods according to the present invention may involve multiple further stages of screening, including selection of test compounds that have a differential effect on target cells in comparison to non-target cells (e.g., a reduced effect on mammalian cells relative to fungal or bacterial cells, or a greater effect on fungal cells relative to bacterial cells or vice versa). This aspect of the invention provides a further screening assay involving (a) contacting a mammalian cell with the metabolic oxidation-reduction indicator dye in the presence and absence of the test compound, and (b) observing the difference in dye signal between cells treated with the test compound and untreated control cells. Test compounds may be alternatively or additionally assayed for ability to kill or inhibit growth of target cells (e.g., fungal cells or bacteria) in vitro using any assays known in the art, including broth or radial diffusion assays, and for toxicity to mammalian cells using any assays known in the art. Suitable compounds may have a 2-fold, 10-fold, 50-fold, 100-fold, or greater separation (selectivity) between antimicrobial activity and mammalian cell activity. Optionally, the in vivo antimicrobial activity of test compounds may also be assayed in any animal models of infection known to those skilled in the art. Such assays include those for in vitro or in vivo oral availability or those for in vivo oral activity as evidenced by activity when administered orally in a comparative survival study.
Another aspect of the invention provides kits for use in conducting the screening methods of the present invention. Such kits may optionally include (a) a metabolic oxidation-reduction indicator dye and (b) a BPI-derived antimicrobial peptide or other BPI protein product suitable for use as a standard (positive control) against which the test compound may be compared.
Other agents that do not exhibit the characteristic xe2x80x9cfingerprint,xe2x80x9d such as amphotericin B, fluconazole, itraconazole or antimycin (for fungal cells) or ciprofloxacin, tetracycline or polymyxin (for bacteria), may be used as an optional negative control.
The present invention also provides novel antimicrobial compounds identified by the screening methods of the present invention.
Yet a further aspect of the invention contemplates the treatment of infections, including fungal or bacterial infections, using compounds identified by the screening methods of the present invention that exhibit the above-described characteristic pattern, other than compounds known in the art (including BPI protein products such as BPI-derived peptides previously known in the art).
Numerous additional aspects and advantages of the invention will become apparent to those skilled in the art upon consideration of the following detailed description of the invention which describes presently prepared embodiments thereof.